Recombinant vaccines are of particular importance in human and veterinary medicine for prophylaxis and therapy of infectious and cancerous diseases. It is the aim of an immunization with a recombinant vaccine to induce a specific immune reaction against a defined antigen, which is effective in prevention or therapy of defined diseases. Known recombinant vaccines are based on recombinant proteins, synthetic peptide fragments, recombinant viruses, or nucleic acids.
Recently, DNA and RNA based vaccines have gained more importance. It has been shown that direct intramuscular injection of plasmid DNA results in a long-lasting expression of the encoded genes (Wolff et al., 1990, Science, 247: 1465-1468). This finding was a major incentive in the field to further investigate the applicability of nucleic acids in immunotherapy. At first, DNA based vaccines against infectious pathogens have been studied (Cox et al., 1993, J. Virol. 67: 5664-5667; Davis et al., 1993, Hum. Mol. Genet. 2: 1847-1851; Ulmer et al., 1993, Science 259: 1745-1749; Wang et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 4156-4160). Furthermore, the applicability of nucleic acids in gene therapy against tumors and for induction of a specific anti-tumor immunity has been studied (Conry et al., 1994, Cancer Res. 54: 1164-1168; Conry et al., 1995, Gene Ther. 2: 59-65; Spooner et al., 1995, Gene Ther. 2: 173-180; Wang et al., 1995, Hum. Gene Ther. 6: 407-418).
Nucleic acid based immunization exhibits a number of advantages. For example, the manufacture of nucleic acid based vaccines is straight forward, relatively inexpensive, and DNA based vaccines are stable for long-term storage. However, in particular, DNA based vaccines exhibit a variety of potential safety risks such as induction of anti-DNA antibodies (Gilkeson et al., 1995, J. Clin. Invest. 95: 1398-1402) and potential integration of the transgene into the host genome. This may lead to the inactivation of cellular genes, an uncontrollable long term expression of the transgene, or oncogenesis, and thus, is generally not applicable for tumor-associated antigens with oncogenic potential such as erb-B2 (Bargmann et al., 1986, Nature 319: 226-230) and p53 (Greenblatt et al., 1994, Cancer Res. 54: 4855-4878).
The use of RNA provides an attractive alternative to circumvent the potential risks of DNA based vaccines. Some of the advantages of RNA based immunization are the transient expression and the non-transforming character. Furthermore, RNA does not have to be transported into the nucleus for the transgene to be expressed, and moreover, cannot be integrated into the host genome. Similar to the injection of DNA (Condon et al., 1996, Nat. Med. 2: 1122-1128; Tang et al., 1992, Nature 356: 152-154), the injection of RNA may result in both a cellular as well as a humoral immune response in vivo (Hoerr et al., 2000, Eur. J. Immunol. 30: 1-7; Ying et al., 1999, Nat. Med. 5: 823-827).
Two different strategies have been pursued for immunotherapy with in vitro transcribed RNA (IVT-RNA), which have both been successfully tested in various animal models. Either the RNA is directly injected into the patient by different immunization routes (Hoerr et al., 2000, Eur. J. Immunol. 30: 1-7) or dendritic cells are transfected with IVT-RNA using conventional transfection methods in vitro and then the transfected dendritic cells are administered to the patient (Heiser et al., 2000, J. Immunol. 164: 5508-5514). It has been shown that immunization with RNA transfected dendritic cells induces antigen-specific cytotoxic T-lymphocytes (CTL) in vitro and in vivo (Su et al., 2003, Cancer Res. 63: 2127-2133; Heiser et al., 2002, J. Clin. Invest. 109: 409-417). Furthermore, it has been shown that direct injection of naked RNA into the lymph nodes of laboratory animals (intranodal injection) leads to uptake of said RNA primarily by immature dendritic cells, probably by a process called macropinocytosis (cf. DE 10 2008 061 522.6). It is assumed that the RNA is translated and the expressed protein is presented on the MHC molecules on the surface of the antigen presenting cells to elicit an immune response.
A major disadvantage of RNA based vaccination is the instability of the RNA in vivo, in particular in the cells of the immune system. Degradation of long-chain RNA from the 5′-end is induced in the cell by the so called “decapping” enzyme Dcp2 which cleaves m7GDP from the RNA chain. Thus, it is assumed that the cleavage occurs between the alpha- and beta-phosphate groups of the RNA-cap.
To inhibit the decapping process and thus increase the stability of RNA in vivo, the effect of phosphorothioate-cap-analogs on the stability of said RNA has been studied. It has been shown that the substitution of an oxygen atom for a sulphur atom at the beta-phosphate group of the 5′-cap results in stabilization against Dcp2. The phosphorothioate modification of the RNA 5′-cap has been combined with an “anti-reverse cap analog” (ARCA) modification that inhibits the reverse integration of the cap into an RNA chain. The resulting cap analog, i.e., m2(7,2′-O)GppspG, was termed beta-S-ARCA (cf. FIG. 1). The replacement of an oxygen atom for a sulphur atom at a bridging phosphate results in phosphorothioate diastereomers which are designated D1 and D2 based on their elution pattern in HPLC. Interestingly, the two diastereomers differ in sensitivity against nucleases. It has been shown that RNA carrying the D2 diastereomer of beta-S-ARCA is almost fully resistant against Dcp2 cleavage (only 6% cleavage compared to RNA which has been synthesized in presence of the unmodified ARCA 5′-cap), whereas RNA with the beta-S-ARCA(D1) 5′-cap exhibits an intermediary sensitivity to Dcp2 cleavage (71% cleavage). Furthermore, the three cap-analogs ARCA, beta-S-ARCA(D1), and beta-S-ARCA(D2) differ in their binding affinity to the eukaryotic translation initiation factor eIF4E. Both of the phosphorothioate cap analogs possess higher affinity for eIF4E than RNAs having conventional 5′-caps. It has further been shown that the increased stability against Dcp2 cleavage correlates with increased protein expression in HC11 cells. In particular, it has been shown that RNAs carrying the beta-S-ARCA(D2) cap are more efficiently translated in HC11 cells than RNAs carrying the beta-S-ARCA(D1) cap.
In summary, RNA is especially well-suited for clinical applications. However, the use of RNA in gene therapy and RNA vaccination is primarily limited by the short half-life of RNA, in particular in the cytoplasm, which results in low and/or insufficient protein expression. Thus, for RNA vaccination it is of particular importance to increase RNA stability in antigen-presenting cells. Since naked RNA injected into the lymph nodes is primarily taken up by immature antigen presenting cells, in particular by immature dendritic cells, it is of particular importance in the context of RNA vaccination to increase the stability of RNA in immature antigen presenting cells. Thus, it is the object of the present invention to provide RNA which is particularly suited for RNA vaccination, i.e., to provide means to particularly stabilize RNA in immature antigen-presenting cells. This technical problem is solved according to the present invention by the subject-matter of the claims.